Differential regulation of cell growth and gene expression by FGF-2 and FGF-4 in pituitary lactotroph GH4 cells

Molecular and Cellular Endocrinology 247 (2006) 183–191

Differential regulation of cell growth and gene expression by FGF-2 and FGF-4 in pituitary lactotroph GH4 cells Twila A. Jackson, David M. Koterwas, Andrew P. Bradford ∗ Program in Reproductive Sciences, Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center at Fitzsimons, Aurora, CO 80045, USA Received 17 May 2005; received in revised form 3 January 2006; accepted 5 January 2006

Abstract Fibroblast growth factors, FGF-2 and FGF-4, are reported to play divergent roles in pituitary differentiation and tumor formation, stimulating cell differentiation or proliferation, respectively. However, mitogenic responses to FGFs have not been extensively characterized and little is known about the molecular mechanisms by which specific FGF isoforms may mediate distinct biological responses. Here we show that FGF-4 but not FGF-2 stimulated DNA synthesis and cell proliferation in GH4 cells. Microarray analyses revealed that FGF-4 induced expression of several oncogenes, growth factor receptors and cell cycle control proteins (e.g. cyclin D3/cdk4, N-myc, c-Raf, insulin and thyroid hormone receptors) while FGF-2 had no effect or down regulated these same genes. These transcriptional responses are consistent with a proliferative and/or tumorigenic role for FGF-4 versus a growth inhibitory effect of FGF-2. FGF-2 and FGF-4 also differentially regulated MAP kinase phosphorylation, which may underlie their isoform-specific effects on cell growth and gene expression. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Fibroblast growth factors; Pituitary; Micro-array; Gene regulation

1. Introduction Fibroblast growth factors (FGFs) are members of a family of polypeptides that play a critical role in development, embryogenesis, angiogenesis and in the regulation of growth, differentiation, motility and survival of cells (Ornitz and Itoh, 2001; Powers et al., 2000). Several FGFs have been identified as oncogenes (Friesel and Maciag, 1995; Mason, 1994) and FGFs and their receptors have been implicated in the formation and progression of tumors in a variety of tissues including pituitary, breast, prostate, ovary, skin and liver (Birnbaum et al., 1991; Leung et al., 1996; Shimon et al., 1998). Several lines of evidence implicate FGFs in pituitary tumorigenesis. FGF-2 was originally identified in extracts of pituitary and is abundant in pituitary cells (Gospodarowicz, 1975). FGF-2 increases prolactin (PRL) secretion from normal pituitary cells and cultured adenomas (Atkin et al., 1993; Baird et al., 1985) and stimulates differentiation of PRL secreting pituitary lactotrophs (Porter et al., 1994). Furthermore, elevated plasma levels of FGF-2 are present

∗

Corresponding author. Tel.: +1 303 724 3507; fax: +1 303 724 3512. E-mail address: [email protected] (A.P. Bradford).

0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2006.01.002

in patients with multiple endocrine neoplasia type 1 (Zimering et al., 1990). The oncogene FGF-4 is expressed in human prolactinomas (Gonsky et al., 1991) and activates both prolactin transcription and secretion in rat GH4 pituitary cells (Schweppe et al., 1997; Shimon et al., 1996). GH4 cells engineered to stably express FGF-4 resulted in the formation of highly aggressive, invasive tumors upon subcutaneous injection into rats (Shimon et al., 1996). Finally, pituitary adenomas exhibit altered FGF receptor subtype and isoform expression (Asghar Abbass et al., 1997; Ezzat et al., 2002). Thus, FGFs may play a critical role in the development and pathogenesis of pituitary prolactinomas, but the mechanism of action and targets of the FGF signaling pathway in pituitary cells remain to be fully elucidated. A critical outstanding question in the field of FGF signal transduction is to elucidate the pathways and molecular mechanisms by which cell specific and FGF isoform specific effects on the range of biological responses are mediated. GH4 rat pituitary tumor cells provide an excellent, physiologically relevant system in which to define and characterize mediators of FGF signaling and the role of FGFs in regulation of cell growth and gene expression. These differentiated neuroendocrine cells express the phenotypic markers PRL and GH, under control of the POU-homeodomain transcription factor

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GHF-1/Pit-1 (Gutierrez-Hartmann, 1994), and maintain normal hormonal and growth factor responses (Gourdji and Laverriere, 1994). We have used this system to characterize FGF regulation of PRL gene expression, identifying FGF response elements in the rPRL promoter and defining critical elements of the FGF signal transduction pathway (Jackson et al., 2001, 2003; Schweppe et al., 1997). Here we describe differential responses of FGF-2 and FGF-4, with respect to mitogenesis, cell cycle and gene regulation, characterize the expression and autophosphorylation of FGFRs in GH4 cells and describe differences in the timecourse of FGF-2 and FGF-4 induced activation of MAP kinase, that may underlie isoform specific FGF responses.

in the S- and G2/M-phase fractions was combined to serve as a proliferative index.

2. Materials and methods

2.6. Reverse transcription and PCR

2.1. Cell culture and transfections GH4T2 rat pituitary tumor cells were grown in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Rockville, MD) supplemented with 12.5% horse serum and 2.5% fetal calf serum, referred to as full serum (Gibco, Rockville, MD) and 50 ␮g/ml penicillin and streptomycin (Gibco, Rockville, MD). Cells were maintained at 37 ◦ C in 5% CO2 . Cells were transfected by electroporation and assayed for luciferase and ␤-galactosidase as described previously (Jackson et al., 2001).

RNA was prepared using the Quiagen Rneasy Kit as described above (Qiagen) and was reverse transcribed using the Advantage RT-for-PCR Kit (Clontech) as per the manufacturer’s protocol. Briefly, 1.0 ␮g of total RNA was incubated with dNTPs, Rnase inhibitor and MMLV reverse transcriptase (42 ◦ C × 1 h). Reverse transcriptase reaction and Dnase activity were stopped by heating to 94 ◦ C × 5 min. One-tenth of the volume of cDNA was used as the template for a standard PCR reaction (0.5 ␮M each primer, 200 ␮M each dNTPs, 1.5 mM MgCl2 , 1 u Taq DNA polymerase in a 50 ␮L final volume.) PCR reaction was 95 ◦ C × 30 s, 55 ◦ C × 30 s, and 72 ◦ C × 1 min.

2.2. Cell growth assay

2.7. Western blotting and immunoprecipitation

Cells were initially seeded into 60 mm dishes at a density of 3.0 × 105 in 3.0 ml of full serum media and allowed to plate down overnight. For growth assays, cells were starved for 16 h in serum free DMEM, washed three times with PBS and then stimulated with 10 ng/ml FGFs in serum free media. Cells were harvested and dispersed in PBS containing 3 mM EDTA and counted using a hemocytometer. Each experimental point was done in triplicate and experiments were performed three times.

A modification of the method described in McClain et al. (1995) was used. Cells were serum starved overnight and incubated for 24 h ±FGFs (10 ng/ml) as indicated. [3H]thymidine (0.5 ␮Ci) was added 12 h prior to harvest. Cells were rinsed twice with 5 ml of chilled phosphate-buffered saline, once with 1 ml of methanol, twice with 5 ml of chilled 5% trichloroacetic acid (w/v), followed by 5 ml of ethanol at 4 ◦ C. The cells were then dissolved in 0.5 ml of 1N NaOH and neutralized with an equal volume of 1N HCl. Aliquots were removed for liquid scintillation counting and protein determination by the Bradford method (Bradford, 1976) using BSA as the standard. Results are presented as percentage of the total thymidine added incorporated into DNA, normalized to cell number or protein.

FGF Receptor isoform specific antibodies; FGFR1 (Flg H-76), FGFR2 (Bek C-17), FGFR3 (C-15) and FGFR4 (C-16), antibodies to cyclin D3 (H-292), cdk4 (C-22), cdk2 (H-298), p21 (C-19) were obtained from Santa Cruz Biotechnology, CA. GH4T2 cells were serum starved overnight and treated with 10 ng/ml FGF-2 or FGF-4 or the equivalent volume of diluent for the indicated times. Cells (107 ) were washed in cold PBS and harvested in 500 ␮l RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, Complete Protease Inhibitor Cocktail (Boehringer Mannheim, Indianapolis, IN)). Equal amounts of protein (50–100 ␮g), as determined by the Pierce Mini BCA protein assay (Pierce, Rockford, IL), were resolved by electrophoresis on 10% polyacrylamide-SDS gels and transferred to an Immobilon-P membrane (Millipore, Bedford, MA) according to the manufacturer’s protocol. Membranes were blocked 1 h at room temperature or overnight at 4 ◦ C in blocking buffer (5% nonfat dry milk in Tris buffered saline (TBS), 0.1% Tween 20) and subsequently incubated with primary antibodies as indicated (Santa Cruz, Santa Cruz, CA) according to the manufacturer’s protocol (1:500 in blocking buffer, 1 h at room temperature). Protein was detected using the Super Signal chemiluminescence assay (Pierce) according to the manufacturer’s protocol. Where indicated, membranes were stripped of antibody for 30 min at 50 ◦ C, according to the manufacturer’s protocol, and re-probed as described above. Immunoprecipitations were performed using ‘Catch and Release’ columns (Upstate Waltham, MA) according to the manufacturers instructions.

2.4. Flow cytometry

2.8. Statistical analysis

GH4 cells were synchronized by treatment with 1 ␮g/ml aphidicolin (Qian et al., 1998) or 2 mM thymidine (Smits et al., 2000) for 24 h as indicated in the figure legends. FGF-2 or FGF-4 (10 ng/ml) were added as indicated and cells incubated in fresh serum free media for an additional 24 h. GH4 cells were harvested using trypsin for 5 min followed by the addition of trypsin inhibitor, pelleted and resuspended at 106 cells/ml in PBS. Cells were then dispersed by sequential passage through 18, 20 and 22 gauge needles and pelleted for 5 min, 1000 × g in a microfuge. Cells were stained with Krishan stain and analyzed by flow cytometry. Flow cytometry was carried out in the UCHSC Cancer Center core facility on an Epics XL-MCL flow cytometer (Coulter Electronics, Hialeah, FL). The percentage of cells in G1, S or G2/M phases of the cell cycle was determined using the ModFit LT analysis program (Veritey Software House, Topsham, ME). The percentage of cells

Values shown in figures are given as the mean ± standard deviation and N indicates number of observations. The data were analyzed using a paired Students T-test. P-values of <0.05 were considered significant.

2.3. Thymidine incorporation into DNA

2.5. RNA isolation and microarray analysis Total RNA was isolated from ∼20 × 106 GH4T2 pituitary cells using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Using the Clontech Atlas Pure Total RNA Labeling System (Clontech, Palo Alto, CA), 40 ug of total RNA were enriched for poly-A+ and used for probe synthesis (Atlas Pure Total RNA Labeling System User Manual (Clontech), Section VII). Probes were purified using Clontech NucleoSpin Extraction Spin Columns using the protocol in Atlas cDNA Expression Arrays User manual, section VI.C (Clontech). Parallel blots were probed using the Atlas cDNA Expression Arrays User manual protocol.

3. Results FGF-2 and FGF-4 were selected for these studies since FGF-2 has been shown to regulate lactotroph differentiation and prolactin secretion (Atkin et al., 1993; Baird et al., 1985; Porter et al., 1994), whereas the oncogenic FGF-4 has been implicated in the development of pituitary lactotroph tumors (Gonsky et

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Fig. 1. Expression and phosphorylation of FGF receptors in GH4 cells. (A) GH4 whole cell extracts (100 ␮g) separated by SDS PAGE were probed with the indicated anti-FGFR antibodies (Santa Cruz Biotechnology, CA) as described in Section 2. (B) GH4 cells were serum starved overnight and treated ±FGF-2, FGF-4 (10 ng/ml) or diluent (C) for 30 min as indicated. Cell extracts (500 ␮g) were immunoprecipitated with anti-FGFR antibodies as indicated and probed with antiphosphotyrosine antibody (PY-100, Cell Signaling).

al., 1991; Shimon et al., 1996). Thus, FGF-2 is reported to promote the differentiated lactotroph phenotype, while FGF-4 is mitogenic (Gonsky et al., 1991; Shimon et al., 1996; Wada et al., 1997; Schonbrunn et al., 1980; Schonbrunn and Tashjian, 1980). 3.1. Expression and phosphorylation of FGF receptors Pituitary neuroendocrine cells have been shown to express FGFR-1 (Gonzalez et al., 1994; Wada et al., 1997) and FGFR4 (Yu et al., 2002; Ezzat et al., 2004), but the exact complement of FGF receptors expressed on GH4 cells was not known. Using Western blotting with FGFR isoform specific antibodies (Santa Cruz Biotechnology) we showed that GH4 cells express all four functional FGF receptors (Fig. 1A). These results were confirmed by reverse transcription polymerase chain reaction (RTPCR) using a set of primers designed to specifically amplify FGFRs 1-4 (data not shown). As shown in Fig. 1B, treatment of GH4 cells with FGF-2 or FGF-4 resulted in tyrosine phosphorylation of FGFR1 and FGFR3 consistent with the reported overlapping binding specificities of these two ligands. We also observed moderate FGF-induced phosphorylation of FGFR4, while FGFR2 did not undergo tyrosine phosphorylation in response to either FGF-2 or FGF-4 (Fig. 1B). Time course and extent of autophosphorylation of FGFRs did not show any significant, reproducible differences with respect to FGF-2 or FGF-4 treatment of GH4 cells. 3.2. Mitogenic effects of FGF-2 and FGF-4 Proliferative responses to FGFs in pituitary cells have not been extensively characterized. Evidence suggests that FGF2 and FGF-4 may exert opposing effects on proliferation and mitogenesis in pituitary cells, inhibiting or promoting growth, respectively (Schonbrunn et al., 1980; Schonbrunn and Tashjian, 1980; Black et al., 1990; Inoue et al., 1991; Prysor-Jones et al., 1989; Gonsky et al., 1991; Shimon et al., 1996). Based upon these observations, we examined the effects of FGF-2 and FGF4 on cell number, DNA synthesis and cell cycle progression in rat pituitary cell lines (Fig. 2). FGF-4 treatment of GH4T2 cells stimulated growth (Fig. 2A) and [3 H] thymidine incorporation (Fig. 2B), whereas incubation with FGF-2 resulted in no

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significant change in cell number and a small but statistically significant reduction in thymidine uptake. FGF-4 also stimulated entry into the cell cycle, increasing the fraction of cells in the S and G2/M phases (Fig. 2C) based upon DNA content assessed by Fluorescence activated cell sorting (FACS). A corresponding reduction in the number of cells in G1 was observed (not shown). In contrast, FGF-2 did not affect cell cycle progression in GH4 cells. Similar results were obtained with GH3 and GH4C1 cells (data not shown). FACS analysis of control or FGF treated GH4 cells did not show a sub G1 DNA peak characteristic of cell undergoing apoptosis. Thus, FGF-4 but not FGF-2 specifically promotes proliferation of GH4 cells. In contrast, FGF-2 and FGF-4 were equipotent activators of PRL transcription, both isoforms enhancing rPRL promoter activity approximately 3.5-fold (Fig. 2D). Hence the observed differential cell growth responses are not attributable to differences in the bioactivity of FGF-2 and FGF-4 in this system. We have previously shown that FGF stimulation of PRL promoter activity is dependent on MAP kinase activation (Jackson et al., 2003, 2001). To determine the functional role of MAPK in FGF-4 induced mitogenesis, GH4 cells were treated ± FGF-4 in the presence or absence of the MEK inhibitor PD98059. As shown in Fig. 3, PD98059 pre-treatment alone had no significant effect, but the inhibitor completely blocked FGF-4-induced tritiated thymidine uptake. These data indicate that FGF-4 dependent mitogenesis in GH4 cells also requires activation of MAPK. 3.3. Analysis of FGF-2 and FGF-4 induced changes in gene expression To further characterize transcriptional regulation by FGF-2 and FGF-4 in GH4 cells and to investigate putative differential regulation of gene expression by FGF-2 and FGF-4, we used rat cDNA arrays to identify isoform specific FGF-regulated target genes (Table 1). GH4 cells were serum starved for 12 h and then treated with FGF-2, FGF-4 (10 ng/ml) or diluent for 8 h. This time point was selected to allow sufficient time to visualize changes in transcription but still focus on direct effects of FGFs. Cells were washed with cold PBS and harvested for RNA using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s directions. Clontech Atlas Pure Total RNA Labeling System was used to label 50 ␮g total RNA from each treatment condition and the labeled probes were used to screen Clontech Atlas Rat 1.2 Arrays (Clontech) according to the manufacturers protocols. Arrays were analyzed with Atlas Image Software (Clontech). Overall, FGF-2 treatment down-regulated 11 genes and increased expression of 15. In response to FGF-4, 26 genes were up regulated and 6 down regulated. The results, shown in Table 1, represent genes exhibiting consistent, significant changes over background in three independent experiments. Based on changes in the level of prolactin mRNA, which we have previously shown to be increased by FGF-2 and FGF-4 (Jackson et al., 2003, 2001), consistent, reproducible changes in expression of greater than 1.5-fold, were considered significant. Interestingly, a number of oncogenes, growth factor receptors, signaling proteins and regulators of the cell cycle were differentially regulated by FGFs 2 and 4. In particular, expression

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Fig. 2. Differential mitogenic effects of FGF-2 and FGF-4. GH4 cells were serum starved overnight and incubated for 24 h ±FGFs (10 ng/ml) as indicated. Results are expressed as mean ± S.D. * p < 0.05. (A) Cells were harvested in PBS containing 3 mM EDTA and counted using a hemocytometer, N = 18. (B) 0.5 ␮Ci 3 H thymidine was added 12 h prior to harvest and incorporation determined as described in the text, N = 12. (C) Cells were stained with propidium iodide and analyzed for DNA content by flow cytometry, as in materials and methods. The percent cells in the proliferative fraction (S + G2/M) is shown, N = 18. Note scale begins at 20. (D) Effects of FGFs on the rPRL promoter. GH4 cells were transiently transfected with 3 ␮g pA3rPRLluc and 0.3 ␮g pCMV␤. Luciferase activity was assayed and normalized to ␤-galactosidase, N = 6.

of several cell cycle related genes that regulate cell cycle entry and progression (Johnson and Walker, 1999; Roberts et al., 1994; Sherr and Roberts, 1999; Vidal and Koff, 2000) was down-regulated in response to FGF-2 and up-regulated by FGF4 treatment. Cyclin D3 was selectively increased by FGF-4 and expression of the cyclin dependent kinases Cdk4 and cdc2 was decreased by 50–60% by FGF-2, but up-regulated twoto three-fold by FGF-4. Cyclin dependent kinase 5, which is not thought to play a role in cell cycle regulation (Hisanaga and Saito, 2003), was not differentially regulated by FGF-2 and FGF-4, illustrating the selective effect of these isoforms on cell cycle related gene expression. Divergent FGF-2/FGF-4 responses were also observed for the proto-oncogenes ras, raf and myc, as mRNA levels were reduced by FGF-2 treatment and increased in response to FGF-4. We also observed FGF-4 induction of receptors for insulin and thyroid hormone, which are known to stimulate growth of primary pituitary lactotrophs and tumor cells (Stahl et al., 1999; Barrera-Hernandez et al., 1999; Kawashima et al., 2000; Yamakawa and Arita, 2004; Suzuki et al., 1999). In contrast, FGF-2 treatment down-regulated insulin

and thyroid hormone receptor ␤ expression. Consistent with previous reports in GH3 cells induced to proliferate with estrogen (Smith et al., 2004), FGF-4 also selectively increased expression of the LDL receptor. In general, these changes in gene expression profiles are consistent with a proliferative and/or tumorigenic role for FGF-4 versus a growth inhibitory or differentiative effect of FGF-2. To confirm FGF dependent, isoform-specific changes in gene expression, we used RTPCR using specific primer sets (Clonetech) as described previously (Dai et al., 2002). Ethidium bromide stained PCR products were quantitated by scanning densitometry (Kodak EDAS 290) and normalized to ␤-actin as a control (Fig. 4). FGF-4 treatment enhanced expression of cyclin D3, and the cyclin D3 associated kinase cdk4. FGF-4 also increased mRNA levels of cdc2 (cdk1) and the proliferation marker p55cdc. In contrast, FGF-2 down regulated expression of all of these genes except cdc2, relative to control levels (Fig. 4). Based upon these results and the specific mitogenic effects of FGF-4, we examined the effects of FGF-2 and FGF-4 on the protein levels of cyclin dependent kinases and cell cycle inhibitors

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Table 1 Isoform specific regulation of gene expression by FGF-2 and FGF-4

Fig. 3. FGF-4 induced DNA synthesis is dependent on MAP kinase. GH4 cells were serum starved overnight and pretreated for 30 min with PD98059 (10 ␮M) or DMSO. FGF-4 (10 ng/ml) was added and tritiated thymidine incorporation measured as described in Section 2. Results are mean ± S.D. of triplicate observations. A representative experiment is depicted. Similar results were obtained in four independent experiments.

(Fig. 5). Western blot analysis of FGF treated GH4 cell extracts shows that cyclin D3 protein was increased approximately threefold by FGF-4 but only slightly elevated (1.4-fold) by FGF-2. A modest increase in cdk4 levels by FGF-4 was also observed. Similarly, FGF-4 increased expression of cdk2 whereas FGF-2 treatment decreased cdk2 protein levels. In contrast, expression of the cdk inhibitor p21 was increased by FGF-2 and not affected by FGF-4 (Fig. 5). No changes in the level of actin in response to FGFs were observed. Thus, consistent with their divergent effects on cell growth and DNA synthesis and the observed changes in gene transcription, FGF-2 and FGF-4 appear to differentially regulate expression of factors promoting and inhibiting cell cycle progression.

FGF-2

FGF-4

Transcription factors Id2; DNA-binding protein inhibitor Id3; DNA-binding protein inhibitor

2.3 2.3

1.9 3.4

Cell cycle regulators G1/S-specific cyclin D3 (CCND3) P55cdc; cell division control protein 20 Cyclin-dependent kinase 4 (CDK4) Cyclin-dependent kinase 5 (CDK5) Cell division control protein 2 (Cdc2)

1.0 0.4 0.5 1.9 0.2

1.9 1.9 1.8 2.1 3.0

Proto-oncogenes A-raf proto-oncogene c-K-ras 2b proto-oncogene c-raf proto-oncogene; raf-1 N-myc proto-oncogene

1.0 0.4 0.4 0.6

2.2 2.1 2.5 1.8

PKC regulators 14-3-3 zeta/delta RAC PK alpha

0.8 1.0

2.0 2.4

Others Insulin receptor Thyroid hormone ␤ receptor Neuronal pentraxin receptor IBP1 IBP2 LDL

0.2 0.2 2.2 0.3 0.5 1.0

3.8 2.8 3.2 2.9 2.1 2.1

Prolactin Na+/K+ ATPase ␣ 1

1.6 2.6

2.1 2.2

GH4 cells were treated ±FGFs (10 ng/ml) or diluent (control) for 8 h. RNA was isolated and used to probe Atlas cDNA arrays (Clontech) as described in the text. Results are the mean fold changes relative to control cells from three independent experiments.

3.4. FGF-2 and FGF-4 differentially activate MAP kinase To begin to elucidate the mechanism of these isoform-specific FGF responses, we examined the ability of FGF-2 and FGF-4 to activate MAPK in GH4 cells (Fig. 6A). While both FGF-2 and FGF-4 induced MAPK phosphorylation and activation, the

Fig. 4. Analysis of FGF dependent gene expression by RTPCR. GH4 cells (5 × 106 ) were serum starved overnight and treated with 10 ng/ml FGF-2 or FGF-4 for 8 h. (A) Total RNA was isolated, reverse transcribed, and used as the template for PCR reactions using gene specific primer pairs. After 25 cycles, the PCR reactions were resolved on a 1% agarose gel and stained with ethidium bromide. (B) Band intensity was analyzed using Kodak EDAS with Image Quant software. Data were normalized to ␤-actin. Results shown are the mean ± S.D. of three independent experiments. * P < 0.05.

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Fig. 5. Effects of FGFs on expression of cell cycle related proteins. GH4 cells were serum starved overnight and treated ±FGFs for 24 h. Whole cell extracts (50 ␮g) were probed with the indicated antibodies (Santa Cruz Biotechnology, CA) and developed by ECL (Pierce) according to the manufacturers directions. Blots were reprobed with antibody to actin (Sigma). Immunoblots were scanned using a Bio-Rad Gel Doc System and analyzed using Discovery Series Quantity One Software (Bio-Rad, Hercules, CA). Band intensities were normalized to each corresponding actin level and results expressed as fold increase relative to control. Data are mean ± S.D. of five experiments, * P < 0.05.

time course and duration of the responses differed markedly (Fig. 6B). Treatment with FGF-4 resulted in rapid transient phosphorylation of MAPK reaching a peak between 10–15 min and subsequently returning to basal levels within 20–30 min. In contrast, FGF-2 induced a rapid onset of MAPK phosphorylation, with a gradual increase in activity reaching a maximum at approximately 30 min. FGF-2 stimulated MAPK phosphorylation was also sustained for 45–60 min, significantly longer than that observed for FGF-4, returning to basal levels within 2 h (not shown). Thus, FGF-2 and FGF-4 exhibited distinct effects on the onset, magnitude and duration of MAPK activity in GH4 cells, which may underlie the observed differential effects of these isoforms on cell growth and gene expression.

4. Discussion

Fig. 6. Time course of activation of MAP kinase by FGF-2 and FGF-4. (A) GH4 cells were plated in serum free media and treated ±FGF-2, FGF-4 (10 ng/ml) or diluent (Control). At the indicated times, cells were harvested and extracts probed with anti-phosphoMAPK antibody (Cell Signaling). Blots were stripped and reprobed with total MAPK antibody (Cell Signaling). A representative chemiluminescence exposure is shown. (B) Immunoblots were scanned using a Bio-Rad Gel Doc System and analyzed using Discovery Series Quantity One Software (Bio-Rad). Phosphorylated MAPK is expressed as a percentage of the total kinase. Results are the mean ± S.D. of five experiments.

FGF-2 and FGF-4 are reported to exert distinct effects on cell growth and tumoral progression in the pituitary (Atkin et al., 1993; Black et al., 1990; Gonsky et al., 1991; Inoue et al., 1991; Norlin et al., 2000; Prysor-Jones et al., 1989; Shimon et al., 1996; Wada et al., 1997; Schonbrunn et al., 1980; Schonbrunn and Tashjian, 1980). Previously, FGF-2 has been shown to inhibit growth and DNA synthesis in rat pituitary cells (Black et al., 1990; Gonsky et al., 1991; Inoue et al., 1991; Wada et al., 1997) and to slow growth of primary rat and human pituitary cells in culture (Schonbrunn et al., 1980; Schonbrunn and Tashjian, 1980; Inoue et al., 1991; Prysor-Jones et al., 1989). Stable over expression of FGF-2 in GH3 cells had no apparent effects on growth rate (Wada et al., 1997) and FGF-2 failed to stimulate proliferation of prolactin secreting adenomas (Atkin et al., 1993). In addition, exposure of dorsal anterior pituitary explants to FGF-2 reduced both the number of Pit-1 positive cells and expression levels of Pit-1, suggesting that FGF-2 exerts a negative control on the generation of Pit-1 positive progenitor cells in the anterior pituitary (Norlin et al., 2000). In contrast, treatment of GH4 cells with FGF-4, or stable over expression of FGF-4 in this cell type, increased cell proliferation and formed rapidly growing invasive tumors (Gonsky et al., 1991; Shimon et al., 1996).

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Consistent with these reports, our results (Fig. 2) demonstrate that FGF-4 increases cell growth and DNA synthesis in GH4 and related pituitary cell lines, whereas FGF-2 had no effect on cell number or cell-cycle progression and showed a small but statistically significant reduction in DNA synthesis. In addition, we have shown that FGF-2 and FGF-4 exhibit distinct profiles of transcriptional regulation in GH4 cells. In accordance with the reported divergent effects of FGF-2 and FGF-4 on pituitary cell growth and tumorigenesis, expression of genes associated with proliferation was generally increased by FGF-4 and decreased by FGF-2. (Table 1, Figs. 4 and 5). The magnitude of these changes is small (two- to three-fold) but reproducible and consistent with the effects of FGFs on PRL mRNA (Jackson et al., 2001). Similar levels of FGF-4 induced, growth associated gene expression were observed in mouse fibroblasts (Guthridge et al., 1996). Moreover, for those genes that are down regulated by FGF-2 and up regulated by FGF-4, the net change in expression is in the order of 3–19-fold. One of the genes divergently regulated by FGF-2 and FGF4 was N-myc, a homolog of c-myc, expressed in tumor cells with neuronal characteristics (Thomas et al., 2004). While the related c-myc oncogene is over expressed in pituitary tumors (Woloschak and Roberts, 1994; Pei, 2001; Suhardja et al., 1999) and correlates with cell proliferation (Burdman et al., 2001; Fornas et al., 2000; Fujimoto et al., 2004; Mukdsi et al., 2004), the role of N-myc in pituitary tumorigenesis is unclear. Similarly, there are no reports of 14-3-3 proteins in pituitary tumors. However, 14-3-3 proteins are known modulators of cell proliferation, regulators of PKC and Raf signal transduction (Aitken, 1996) and may play a role in the development and progression of cancers (Wilker and Yaffe, 2004). Moreover, the selective induction of 14-3-3 delta and the PKC binding protein (RACK) expression by FGF-4 (Table 1) are consistent with our previous results showing that FGF signaling in GH4 cells is mediated by PKC (Jackson et al., 2001). The D type cyclins serve as growth factor sensors, linking mitogenic stimuli to the cell cycle machinery. Cyclin D transcription, and assembly of cdk/cyclin D complexes are mitogendependent (Sherr and Roberts, 1999). Our results (Figs. 4 and 5 and Table 1) using array analysis, semi-quantitative RTPCR and Western blotting, show that FGF-4 up regulates levels of cyclin D3 mRNA and protein, whereas FGF-2 treatment decreased cyclin D3 transcript levels. We also observed FGF-4 specific up regulation of the cyclin D associated kinase cdk4. Cyclin D3 is the most widely expressed of the D type cyclins and is essential for cell cycle progression but is the least well characterized with respect to its effects on cell growth and oncogenesis (Bartkova et al., 1998). Endocrine tumors are frequently strongly cyclin D3 positive (Depoortere et al., 1998; Doglioni et al., 1998; Ebert et al., 2001; Saeger et al., 2001). Cyclin D3 is the most abundant D-type cyclin in prolactin secreting rat pituitary cells and specific changes in cyclin D3-bound cdk4 were observed in rat pituitary tumors correlating with levels of Rb protein (Chun et al., 1998). Similarly, thyroid hormone (T3) induced proliferation of pituitary GC cells by increasing levels and activity of cyclin D/E associated kinases (Barrera-Hernandez et al., 1999). Thus, consistent with our observations in prolactinoma cells,

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evidence suggests that cyclin D3 is a critical component in the pathogenesis of endocrine tumors. Effects of FGF-4 on cyclins, cdks and cell cycle progression have not been described. However, FGF-2 has been shown to modulate cyclin, cdk and CKI levels in cell lines derived from breast (Wang et al., 1997), chondrocytes (Aikawa et al., 2001) and neuroepithelium (Smits et al., 2000), resulting in growth inhibition. The inhibitory effects of FGF-2 in MCF-7 breast cancer cells and rat chondrosarcoma cells are mediated by increases in the levels of p21 and subsequent inactivation of cyclin E/cdk complexes (Aikawa et al., 2001; Wang et al., 1997), whereas growth inhibition of SK-N-MC neuroepithelioma cells by FGF-2 is mediated by blocking activation of the mitotic cdk, cdc2 (Smits et al., 2000). In accordance with these results, we also observed FGF-2 increased p21 protein (Fig. 5) and decreased expression of cdc2 (Table 1), while FGF-4 had no effect on p21 levels and increased cdc2 mRNA. Effects of FGFs may be modulated by the level and duration of activation of distinct FGFRs (LaVallee et al., 1998; Raffioni et al., 1999; Rusnati et al., 1996) or interaction with and activation of diverse downstream signaling pathways (Choi et al., 2001; Hadari et al., 2001; Kanai et al., 1997; Maher, 1999; Ong et al., 2000, 2001; Shaoul et al., 1995; Vainikka et al., 1994). Few studies have addressed diverse responses mediated by different members of the FGF family of ligands. Our results demonstrate that, despite their similar induction of rPRL promoter activity (Jackson et al., 2003, 2001; Schweppe et al., 1997), FGF-2 and FGF-4 exhibit divergent effects on regulation of pituitary cell growth and gene expression (Figs. 4 and 5 and Table 1). Both FGF-2 and FGF-4 induced similar phosphorylation of FGFR1, 3 and 4 in GH4 cells (Fig. 1B), suggesting that isoform specific responses are not a result of selective FGFR activation. FGFRs have been shown to induce distinct levels and duration of Ras-dependent and Ras-independent signaling pathways controlling cell proliferation, differentiation and gene expression (Cailliau et al., 2001; Choi et al., 2001; Presta et al., 1991; Maher, 1999). FGFs 1 and 2 were shown to differentially activate MAPK pathways induced by FGFR4 in Xenopus oocytes (Cailliau et al., 2001) suggesting that FGF isoforms can elicit distinct responses from the same receptor. Consistent with these reports, we observed markedly different responses to FGF-2 and FGF-4 with respect to activation of MAPK. FGF-4 treatment resulted in rapid but transient MAPK phosphorylation, while FGF-2 induced prolonged MAPK activation (Fig. 6). Given that FGF-4 mitogenic responses in GH4 cells are dependent on MAPK (Fig. 3), we propose that the divergent effects of FGF-2 and FGF-4 on cell growth and gene expression may be in part a reflection of their distinct isoform-specific MAPK activation profiles. In summary, we have shown that FGF-2 and FGF-4 exhibit distinct patterns of neuroendocrine gene regulation, consistent with their reported divergent effects on lactotroph proliferation and pituitary tumorigenesis. FGF-2 and 4 activated a similar complement of FGF receptors in GH4 cells and thus, their isoform-specific responses are likely a function of distinct effects on downstream signaling pathways.

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